The multifunctional oncoprotein MDM2 plays critical roles in cancer initiation, progression and the development of resistance to therapy. We studied pediatric cancer patients, including those with acute lymphoblastic leukemia (ALL) and neuroblastoma (NB), finding that patients with a poor prognosis commonly had cancer cells that expressed constitutively high levels of MDM2. An important function of MDM2 is the ubiquitination (as an E3 ligase) and degradation of the tumor suppressor p53, due to interactions with DAXX and HAUSP. Interestingly, disruption of these interactions can result in self-ubiquitination of MDM2 and p53 activation. Thus, we believe targeting MDM2 by disrupting MDM2-DAXX-HAUSP interactions is an innovative approach to develop new therapeutics for MDM2-overexpressing cancer patients. Berberine (BBR), an isoquinoline alkaloid derived from a traditional Chinese medicinal herb, has been shown to have anti-proliferative and pro-apoptotic effects on human cancer. Our preliminary data supports a previously unrecognized mechanism of action for BBR: It downregulates MDM2 by inhibiting DAXX, disrupting MDM2-DAXX-HAUSP interactions. In contrast to the conventional chemotherapeutic drug doxorubicin (Dox) that induces prior p53 activation and a subsequent upregulation of MDM2, we discovered that BBR strongly induced persistent downregulation of MDM2, followed by a steady-state activation of p53, resulting in potent apoptosis of MDM2-overexpressing cancer cells, including those that are Dox-resistant. This proposal is designed to test our central hypothesis that regulation of MDM2 by DAXX inhibition is the major mechanism by which BBR differs from Dox in exerting its anti-cancer effect. The project's long term objectives are: characterization of the critical role BBR plays in the downregulation of MDM2 to overcome chemoresistance and determination of BBR's potential as a therapeutic reagent for refractory cancer patients. The specific aims of this study are: 1) To fully elucidate the cellular/molecular mechanisms by which BBR downregulates MDM2; 2) To establish that downregulation of MDM2 is the primary mechanism by which BBR exerts its anticancer effects; and 3) To assess the ability of BBR to reverse chemoresistance and its potential as a treatment for refractory cancer patients. Successful completion of these specific aims will generate knowledge with respect to the mechanisms of action for BBR prior to future clinical trials of this natural product in pediatric cancer patients, should introduce BBR as a highly useful new drug candidate for overcoming chemoresistance, and also will provide a basic framework for the rational design of other therapeutic approaches targeting the DAXX-MDM2 pathway.